Odn1668

Manufactured by InvivoGen

Sourced in France

ODN1668 is a synthetic oligodeoxynucleotide that contains a CpG motif. It acts as a Toll-like receptor 9 (TLR9) agonist, stimulating the immune system by activating TLR9-expressing cells.

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Lab products found in correlation

One glo ex reagent, by Promega (2 mentions) Odn2088, by InvivoGen (2 mentions) Quanta 250, by Thermo Fisher Scientific (1 mentions) C57bl 6j b6 mice, by Jackson ImmunoResearch (1 mentions) Control odn, by InvivoGen (1 mentions) Pentobarbital i p, by Merck Group (1 mentions) Fei quanta 250, by Thermo Fisher Scientific (1 mentions) Dxr 500, by Bruker (1 mentions) High molecular weight poly 1 c, by InvivoGen (1 mentions) Fsl 1, by InvivoGen (1 mentions) Alexa fluor 488 donkey anti rat igg, by Thermo Fisher Scientific (1 mentions) Alexa fluor 568 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions) Poly 1 c hmw, by InvivoGen (1 mentions) Odn m362, by InvivoGen (1 mentions) Anti lamp 1 1d4b, by Abcam (1 mentions) Anti eea1, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions) Odn1585, by InvivoGen (1 mentions) Anti phospho sapk jnk 9255, by Cell Signaling Technology (1 mentions) Puno mtlr9 ha, by InvivoGen (1 mentions)

20 protocols using odn1668

Proximal Tubule TLR9 Activation Assay

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Mouse kidney proximal tubules were isolated with Percoll density gradient separation as described previously^{55 (link)}. Confluent cells were treated with control oligonucleotides (ODN) or with selective 5 mM TLR9 agonist ligand ODN1668 (InvivoGen, CA) for 3 days as described^{56 (link)}. Some cells were pretreated with MNPs encapsulating ODN2088 (87 ng ODN2088/mg MNPs) 30 min before control ODN or ODN1668 treatment. We then performed caspase 3 and caspase 8 immunoblotting as described^{51 (link)}.

Han S.J., Williams R.M., D’Agati V., Jaimes E.A., Heller D.A, & Lee H.T. (2020). Selective nanoparticle-mediated targeting of renal tubular Toll-like receptor 9 attenuates ischemic acute kidney injury.. Kidney international, 98(1), 76-87.

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Synthesis and Characterization of CPTEG:CPH Nanoparticles

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1,8-bis(p-carboxyphenoxy)-3,6-dioxoctane (CPTEG) and 1,6-bis(p-carboxyphenoxy)hexane (CPH) monomers were synthesized as previously described (34 (link), 47 (link)). 20:80 CPTEG:CPH copolymer was synthesized as previously described (47 (link)). 20:80 CPTEG:CPH nanoparticles containing 2% CpG (ODN 1668, Invivogen, San Diego, CA) and/or 1% RSV prefusion F (DS-Cav1, a gift from Dr. Jason McLellan, University of Texas at Austin, Austin, TX) were synthesized via solid-oil-oil double emulsion (43 (link)). Briefly, copolymers, RSV F, and CpG were dissolved at a polymer concentration of 20 mg/mL in methylene chloride. The solution was sonicated for 30 s and precipitated into chilled pentane. The resulting nanoparticles were collected via vacuum filtration and their morphology and size were characterized using scanning electron microscopy (FEI Quanta 250, FEI, Hillsboro, OR).

Stephens L.M., Ross K.A., Waldstein K.A., Legge K.L., McLellan J.S., Narasimhan B, & Varga S.M. (2021). Prefusion F-based polyanhydride nanovaccine induces both humoral and cell-mediated immunity resulting in long-lasting protection against respiratory syncytial virus. Journal of immunology (Baltimore, Md. : 1950), 206(9), 2122-2134.

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Culturing Leukocytes with CpG Stimuli

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Total leukocyte populations from spleen or blood were cultured at 20°C in L-15 medium supplemented with antibiotics and 5% FCS in 24 or 96-well plates (Nunc). Different stimuli were added to the media and cells were incubated for different time periods depending on specific experiments. The phosphorothioate-modified B class CpG oligodeoxynucleotide (ODN) 1668 (InvivoGen) containing one CpG dinucleotide (CpG) (5′-tccatgaCGttcctgatgct-3′) was used at a final concentration of 5 μM after having determined the optimal concentration based on their positive effect on B cell survival, specifically choosing the concentration that provoked the higher B cell survival after 72 h of incubation (data not shown). The non-CpG ODN 1668 (that contains GpC dinucleotides instead of CpGs) (5′-tccatgaGCttcctgatgct-3′) was used as a negative control (non-CpG) at the same concentration. In some experiments, leukocytes were stimulated with an unlabeled monoclonal antibody (mAb) against trout IgM (clone 1.14, mouse IgG1) (20 (link)) at a final concentration of 10 μg/ml as previously described (5 (link)). Non-stimulated controls were always included.

Simón R., Díaz-Rosales P., Morel E., Martín D., Granja A.G, & Tafalla C. (2019). CpG Oligodeoxynucleotides Modulate Innate and Adaptive Functions of IgM+ B Cells in Rainbow Trout. Frontiers in Immunology, 10, 584.

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Renal Ischemia-Reperfusion Injury in Mice

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After Columbia University Institutional Animal Care and Use Committee approval, 8–10 week old male TLR9 proximal tubule deficient (TLR9^fl/fl PEPCK Cre) mice or control wild type (TLR9^fl/fl) mice weighing 20–25g were anesthetized with pentobarbital i.p. (Sigma, St Louis, MO: 50 mg/kg body weight or to effect). Mice were then subjected to right nephrectomy and 30 min left renal ischemia as described previously (37 , 38 (link)). A separate cohort of TLR9^fl/fl mice or renal tubular TLR9 null mice were pretreated with a selective TLR9 agonist (1 mg/kg ODN-1668 or control ODN, Invivogen, San Diego, CA) and subjected to 20 min renal IR injury. Some mice were treated with vehicle for ODN (saline) before 20 min renal ischemia. To determine the effects of global TLR9 deletion in ischemic AKI, we also subjected TLR9 knockout mice (TLR9KO, C57BL/6J^{Tlr9M7Btlr/Mmjax}) and their wild type controls (C57BL/6J mice (B6)) to 30 min renal IR injury (Jackson Labs, Bar Harbor, ME). For pain management, all mice received 0.5–1 mg/kg s.c. buprenorphine SR prior to surgery. Sham-operated animals underwent anesthesia followed by laparotomy, right nephrectomy, bowel manipulations and wound closure without renal ischemia. Body temperature of all mice were sustained at ~37°C using a surgical heating pad during surgery as well as during recovery from anesthesia.

Han S.J., Li H., Kim M., Shlomchik M.J, & Lee H.T. (2018). Kidney proximal tubular TLR9 exacerbates ischemic acute kidney injury. Journal of immunology (Baltimore, Md. : 1950), 201(3), 1073-1085.

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Synthesis and Characterization of CPTEG:CPH Nanoparticles

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Monomers based on 1,8-bis(p-carboxyphenoxy)-3,6-dioxoctane (CPTEG) and 1,6-bis(p-carboxyphenoxy)hexane (CPH) were synthesized as described previously (30 (link), 31 (link)) Using these monomers, 20:80 CPTEG:CPH copolymer was synthesized using melt polycondensation for ~6 h, as described (31 (link)). The final copolymer composition, purity, and molecular weight of the copolymer were characterized using ¹H HNMR (DXR 500, Bruker, Billerica, MA). Next, 20:80 CPTEG:CPH nanoparticles containing 1% H1 HA, 1% NP, and 2% CpG1668 were synthesized via solid-oil-oil double emulsion (32 (link)). Briefly, HA and NP protein antigens (Sino Biological, Beijing, China) were dialyzed to nanopure water and lyophilized overnight. The 20:80 CPTEG:CPH copolymer, along with HA, NP, and CpG (ODN 1668, Invivogen, San Diego, CA), was dissolved at a polymer concentration of 20 mg/mL in methylene chloride. The solution was sonicated for 30 s and then precipitated into chilled pentane (at a methylene chloride:pentane ration of 1:250). The resulting nanoparticles were collected via vacuum filtration and scanning electron microscopy (FEI Quanta 250, FEI, Hillsboro, OR) was used to characterized morphology and size.

Zacharias Z.R., Ross K.A., Hornick E.E., Goodman J.T., Narasimhan B., Waldschmidt T.J, & Legge K.L. (2018). Polyanhydride Nanovaccine Induces Robust Pulmonary B and T Cell Immunity and Confers Protection Against Homologous and Heterologous Influenza A Virus Infections. Frontiers in Immunology, 9, 1953.

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Peritoneal Macrophage Harvesting and Stimulation

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C57BL/6J WT and MyD88-deficient mice were obtained from the Jackson Laboratory
(Bar Harbor, Maine). Harvesting, culturing, and stimulation of peritoneal macrophages were
described previously (Toshchakov et al., 2005 (link)).
THP-1 cells were differentiated by incubation in the presence of 10 nM PMA in 10%
FCS RPMI-1640 for 3 days. TLR agonists, P3C, P2C, FSL-1, R848, ODN1668, and high molecular
weight poly (I:C) were purchased from InvivoGen (San Diego, CA). Escherichia
coli K235 LPS was phenol-purified. Mouse rTNF was purchased from Biolegend,
Inc.

Piao W., Shirey K.A., Ru L.W., Lai W., Szmacinski H., Snyder G.A., Sundberg E.J., Lakowicz J.R., Vogel S.N, & Toshchakov V.Y. (2015). A decoy peptide that disrupts TIRAP recruitment to TLRs protects in a murine model of influenza. Cell reports, 11(12), 1941-1952.

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Deciphering Immune Response Pathways

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Anti-CD11c (N418), MHC class II (I-A) (NIMR-4), anti-CD40 (HM40-3), Anti-CD80 (16-10A1), Anti-CD86 (GL1) and anti-mTLR9 (M9.D6) were obtained from eBioscience. Anti-β-actin (AC-74) and thioglycolate were from Sigma-Aldrich. Anti-p44/42 MAPK (4695), anti-phospho-p44/42 MAPK (9101), anti-p38 MAPK (9212), anti-phospho-p38 MAPK (9216), anti-IkB-α (9242), anti-phospho-IkBα (2859), anti-SAPK/JNK (9258), anti-HA (2367 and 3724) and anti-phospho-SAPK/JNK (9255) were from Cell Signaling Technology. Anti-EEA1 was from Thermo Fisher Scientific. Anti-LAMP1 (1D4B) was from abcam. Alexa Fluor488 Donkey anti-mouse IgG, Alexa Fluor488 Donkey anti-rat IgG and Alexa Fluor568 Donkey anti-rabbit IgG were from life technologies. Affinity-purified rabbit polyclonal anti-CRAMP Ab which recognizes CRAMP peptide domain was previously generated in our laboratory(38 (link)). ODN 1585, ODN 1668, ODN M362, ODN 1668-FITC, ODN M362-FITC, R848, Poly(I:C) HMW, Pam3CSK4, Zymozan, LPS-EB, pUNO-mTLR9-HA and pSELECT-puro-mcs were from invivogen. DOTAP liposomal transfection reagent (DOTAP) was from Roche applied science. Recombinant mouse CRAMP peptide (mCRAMP) was synthesized by Genemed Synthesis Inc.

Nakagawa Y, & Gallo R.L. (2014). Endogenous intracellular cathelicidin enhances TLR9 activation in dendritic cells and macrophages. Journal of immunology (Baltimore, Md. : 1950), 194(3), 1274-1284.

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Packaging CpG ODN 1668 into VLPs

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Synthetic oligodeoxynucleotide (ODN) 1668 (5′-tccatgacgttcctgatgct-3′) is the B-class unmethylated CpG dinucleotide specific for mouse Toll-like receptor 9 (TLR9), strongly activates B cells but weakly IFN-α secretion. ODN 1668 was obtained from InvivoGen (Toulouse, France) and it was tested here with 183preS1(20–47) VLPs for the packaging. ODN packaging was performed according to the method described in [66 (link)] with the use of RNase and urea. For packaging the following mix was prepared: 50 µg/12,5 µL of VLPs in PBS + 100 µg/10 µL RNaseA (Thermo Fisher Scientific, cat. No R1253) + 50 µL 1 M urea in water + 7 µg/30 µL ODN 1668 in water. The packaging mix was incubated at room temperature overnight.

Dishlers A., Petrovskis I., Skrastina D., Zarina I., Lieknina I., Jansons J., Akopjana I., Zakova J., Ose V, & Sominskaya I. (2023). PreS1 Containing HBc VLPs for the Development of a Combined Therapeutic/Prophylactic Hepatitis B Vaccine. Microorganisms, 11(4), 972.

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Luciferase Assay for TLR9 Signaling

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HEK293T cells stably transfected with a mouse TLR9/NFκB/Luciferase reporter plasmid were treated with different concentrations of the respective ASO or a positive control (ODN1668, Invivogen) for 24 hours. For detection of luciferase activity, ONE-Glo™ EX reagent (Promega) was added to the cells and luminescence was measured on a BMG Clariostar reader.

Shen M., Xie S., Rowicki M., Michel S., Wei Y., Hang X., Wan L., Lu X., Yuan M., Jin J.F., Jaschinski F., Zhou T., Klar R, & Kang Y. (2020). Therapeutic Targeting of Metadherin Suppresses Colorectal and Lung Cancer Progression and Metastasis. Cancer research, 81(4), 1014-1025.

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TLR9-NF-κB Luciferase Reporter Assay

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The Toll-like receptor 9 (TLR9) reporter gene assay was performed as previously described (41 ). Briefly, stably transfected human embryonic kidney cells expressing a mouse TLR9 NF-κB luciferase reporter plasmid were treated with ODN1668 (catalog no.: tlrl-1668; InvivoGen) and ANGPTL4 ASO or Neg-Ctrl ASO at indicated concentrations (5,000, 1,000, 200, 40, 8, and 1.6 nM). Each condition was performed in triplicates. After 24 h, the cell supernatants were removed, and ONE-Glo EX reagent (50 μl, catalog no.: E8110; Promega) was added for cell lysis according to the manufacturer’s instructions. Luminescence was immediately measured at 560 nm. Data are represented as the mean of triplicate wells ± SD.

Deng M., Kutrolli E., Sadewasser A., Michel S., Joibari M.M., Jaschinski F., Olivecrona G., Nilsson S.K, & Kersten S. (2022). ANGPTL4 silencing via antisense oligonucleotides reduces plasma triglycerides and glucose in mice without causing lymphadenopathy. Journal of Lipid Research, 63(7), 100237.

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